Identification and Characterization of Proteins
That Selectively Interact with Isoforms of the
mRNA Binding Protein AUF1 (hnRNP D)

Publication History

Published Online:

2005-06-01

Abstract

The mRNAs that encode certain cytokines and proto-oncogenes frequently contain a typical AUrich motif
that is located in their 3'-untranslated region. The protein AUF1 is the first factor identified that binds to AU
rich regions and mediates the fast degradation of the
target mRNAs. AUF1 exists as four different isoforms
(p37, p40, p42 and p45) that are generated by alternative splicing. The fact that AUF1 does not degrade
mRNA itself had led to the suggestion that other AUF1
interacting proteins might be involved in the process
of selective mRNA degradation. Here we used the
yeast two-hybrid system in order to identify proteins
that bind to AUF1. We detected AUF1 itself, as well as
the ubiquitin-conjugating enzyme E2I and three RNA
binding proteins: NSEP-1, NSAP-1 and IMP-2, as
AUF1 interacting proteins. We confirmed all interactions in vitro and mapped the protein domains that are
involved in the interaction with AUF1. Gelshift assays
with the recombinant purified proteins suggest that
the interacting proteins and AUF1 can bind simultaneously to an AU-rich RNA oligonucleotide. Most interestingly, the AUF1 interacting protein NSEP-1showed
an endoribonuclease activity in vitro. These data suggest the possibility that the identified AUF1 interacting proteins might be involved in the regulation of
mRNA stability mediated by AUF1.